From RNA to Protein

• Translation

• Protein

MB 207 – Molecular Cell Biology

Overall view of Protein Synthesis

The ribosome (ribosomes read messenger RNA and direct the synthesis of

the encoded protein)

• The cellular factory responsible for synthesizing proteins • Consists of various rRNAs and about 50 different proteins (ribosomal proteins): 2/3 RNAs &

1/3 proteins• RNAs instead of proteins with the catalytic activity, proteins are there as to stabilize the RNA

core• Assembled in the nucleolus• Inactive state: exists as two subunits: a large subunit & a small subunit• When the small subunit encounters an mRNA, the process of translation of the mRNA to protein begins. • Contain 4 binding sites: 1 mRNA binding site & 3 tRNA binding sites:

A (aminoacyl) P (Peptidyl) E (Exit) site

Comparison procaryotic and eucaryotic ribosome structures (both have similar structure and function)

S= rate of sedimentation in an ultracentrifuge

Ribosome subunits

Prokaryotes Eukaryotes

Small subunits 30S 40S

Large subunits 50S 60S

Whole ribosome 70S 80S

Svedberg (S): a unit of sedimentation velocity (sedimentation is an ultracentrifuge depends on both the mass and shape of a molecule

Charging the tRNA• tRNA is the translator between mRNA and protein (transfer genetic information to

aa sequence)• tRNA has a specific anticodon and acceptor site• tRNA has a specific charger protein - aminoacyl tRNA synthetases

can only bind to that particular tRNA and attach the correct amino acid to the acceptor site

Energy to make this bond comes from ATPThe genetic code is translated by means of the two adaptors that act one after another:The genetic code is translated by means of the two adaptors that act one after another:1)1) aminoacyl-tRNA synthetase (couples an aa to it’s corresponding tRNA)aminoacyl-tRNA synthetase (couples an aa to it’s corresponding tRNA)2)2) tRNA molecule (anticodons forms bp with codon on the mRNA)tRNA molecule (anticodons forms bp with codon on the mRNA)

codon

anticodon

Translation

• The process that uses the base sequence in mRNA to synthesize a polypeptide with "complementary" amino acid sequence

• The information in mRNA is always read from the 5' to the 3' direction. The polypeptide is synthesized from its amino terminus to its carboxyl terminus.

RNA: 5'----------------------------------3'

polypeptide: H2N-----------------------------COOH

• The translation process occurs at the ribosome in the cytoplasm• Involves the three major classes of RNA: mRNA, tRNA, and rRNA

as well as free amino acids, free energy, and several non-ribosomal protein factors

The ribosomal subunit cycle during protein synthesis

30S subunits with initiation factors

Separate subunits

Pool of free ribosomes

Initiation Elongation Termination

Initiation factors

Initiation requires free ribosome subunits. When ribosomes are released at termination, they dissociate to generate free subunits. Initiation factors are present only on dissociated 30S subunits. When subunits reassociate to give a functional ribosome at initiation, they release the factors.

(Ribosomal subunits assemble and disassemble during each round of protein synthesis in the ribosome cycle)

Translation initiation requires:

Ribosome brought to mRNA

Ribosome properly aligned over start codon

P site of ribosome containing charged tRNA

The initiation process differs significantly in prokaryotes and eukaryotes

Initiation is aided by translation initiation factors

Prokaryotes

Eukaryotes Function

IF1 eIFA Blocks ribosome A site

IF2 eIF, eIF5b Facilitate initiator tRNA binding to the P site of 40S subunit

IF3 eIF3 Prevents ribosomal subunit association

eIF, eIF4A, eIF4E, eIF4G, eIF4B

Prepare mRNA template for ribosome binding

Translation initiation

Bacterial mRNA are often polycistronic – encode several different proteinsEucaryotes mRNA only encode 1 single protein

Translation initiation in Prokaryotes

•mRNA start codon and fMet-tRNA anticodon are aligned to start translation

•Base-pairing of mRNA start codon and fMet-tRNA initiates reaction cascade to form 70S initiation complex:

IF3 dissociates

Large subuntis binds

GTP is hydrolyzed by IF2

IF2 and IF1 dissociate

•Protein synthesis begins

IF1, IF2 and IF3 bind 30S subunits (IF3, then IF2-GTP and IF1)IF1 prevents tRNAs from entering A siteIF2 binds IF1 and guides fMet-tRNA to P siteIF3 prevents association of large subunitShine-Dalgarno sequence base pairs with 16S rRNA on ribosome

Initiation of Translation• The start signal for translation is the codon ATG (AUG), which codes for

methionine (met) (formyl-met for prokaryotes) – all newly synthesized protein has a met at the N-terminal

• A tRNA charged with met is required to bind to the translation start signal• An complex consists of the small ribosomal unit, an initiation factor (eIFs)

and the tRNAmet is formed• The complex bind to the 5’ end of an mRNA (recognize the 5‘ Cap for

eukaryotes or Shine-Dalgarno sequence for prokaryotes) and move forward along the mRNA to search for the initiator codon AUG- Scanning requires energy - ATP hydrolysis- It is not always the first AUG that is recognized as a start codon,

sequences around first AUG might reduce efficiency of initiation so scanning continues to next AUG.

• Initiation factors dissociate with GTP hydrolysis, and the large ribosomal subunit associates with the complex, the initiator tRNA bind at P site

• Protein synthesis is ready to begin

The initiation phase of protein synthesis in eukaryotesThe initiation phase of protein synthesis in eukaryotes43S pre-initiation complex formed by:

•40S subunit

•Initiation factors eIFA, eIF3, eIF5B-GTP and eIF-Met-tRNA-GTP

eFI4F complex (E,G and A) binds to mRNA

43S pre-initiation complex brought to mRNA by eIF4 family of translation initiation factors

Helicase activity of eIF4 family helps 40S subunit seat on the start codon

eIF2 and eIF3 are released from the complex

60S subunit binds 40S subunit and initiation factors are released

Ribosome is now ready to begin protein synthesis

Elongation requires:

Placement of charged tRNA in the A site

Peptide bond formation

Translocation

Elongation factors EF-Tu, EF-G and EF-Ts

Elongation of the New Protein

• A next tRNA carrying an other amino acid is attracted and pairs with the next codon at the A site, the peptide bond is catalysed by a ribosomal protein (peptidyl-transferase) associated with the large ribosomal subunit.

• The result is a transfer of the N-methionine to the second tRNA in the A site which carries now a dipeptide and the initiator tRNA being uncharged in the P site.

• Formation of each peptide bond is energetically favorable because the growing C-terminus has been activated by the covalent attachment of a tRNA molecule

The incorporation of an amino acid into a proteinThe incorporation of an amino acid into a protein

Elongation of the New Protein• The first tRNA is now released (move to E site) and the ribosome shifts so that a

tRNA carrying two amino acids is now in the P site, and leaves the A site unoccupied but with the third codon exposed.

• Translocation: – Ribosome moves to the next codon– Empty tRNA is ejected and the peptidyl-tRNA is shifted from the A site to the

P site– New aminoacyl-tRNA is allowed to enter within the A site– Translocation is catalyzed by the elongation factor EF-G. The G indicates that this factor uses the energy gained from the hydrolysis of

GTP for translocation to occur. – Finally a third tRNA is escorted by EF-Tu to the A site and the anticodon

base pairs specifically with the codon. – This binding triggers the release of the initiator tRNA from the E site and

allows the complex to be ready for another round of peptide bond formation, translocation and codon-anticodon base pairing

– And so on…..

View of the translation cycle

Incorrectly base paired tRNAs preferentially dissociate

The aminoacyl-tRNA is escorted to the A site by the elongation factor EF-Tu

The peptidyl transferase reaction transfer the amino acid from the P site onto the aminoacyl-tRNA in the A site

During translocation:

•Ribosome moves one codon down mRNA

•tRNA with growing protein chain moves into P site

•Spent tRNA moves into E site

•Translocation requires EF-G

Translating an mRNA moleculeTranslating an mRNA molecule

Step 1 An aminoacyl-tRNA bind to a vacant A-site Step 1 An aminoacyl-tRNA bind to a vacant A-site on the ribosome.on the ribosome.Step 2 A new peptide bond is formStep 2 A new peptide bond is formStep 3 The mRNA moves a distance of 3 nts through the Step 3 The mRNA moves a distance of 3 nts through the small –subunit chain, ejecting the spent tRNA small –subunit chain, ejecting the spent tRNA molecule and ‘resetting’ the ribosome so that the molecule and ‘resetting’ the ribosome so that the next incoming aminoacyl-tRNA molecule can bindnext incoming aminoacyl-tRNA molecule can bind

The 3 steps cycle is The 3 steps cycle is repeated over and repeated over and over again over again duringduring

protein synthesis.protein synthesis.

Termination

Stop codon signals termination: UAG, UGA, UAA

Release factors (RF) accomplish termination

Termination is similar in prokaryotes and eukaryotes

Prokaryotes Eukaryotes

•Class I •RF1 recognizes stop codon UAG•RF2 recognizes stop codon UGA•RF1 & 2 recognizestop codon UAA

eRF1 recognizes all stop codons

•Class II •RF3 eRF3

Termination of the Protein Synthesis

• When the ribosome reaches a stop codon, no aminoacyl tRNA binds to the empty A site. This is the ribosomes signal to break into its large and small subunits, releasing the new protein and the mRNA.

• Stop codons are triplets which are not recognized by any tRNA (UAA,

UAG, UGA), but by a protein releasing factor (RF1 or RF2 in prokaryotes, eRF in eukaryotes).

• The factor R binds to the A site forcing the peptidyl transferase to catalyze the addition of water to the peptidyl-tRNA and causes the release of the polypeptide chain

The final phase of protein synthesis

•This release in This release in turn causes the turn causes the complex to complex to dissociate and dissociate and mRNA, tRNA mRNA, tRNA and ribosomal and ribosomal subunits are subunits are freed.  freed.  

Polyribosome

• A single mRNA molecule is not only translated once– As soon as the ribosome

has moved away from the initiation site, another round of initiation can begin

– A single mRNA is often transcribed by many ribosomes at the same time, usually 100 to 200 bases apart from each other

• A group of ribosomes on the same mRNA is called a polyribosome or polysome

• Many proteins can be made in a given time

Protein folding

• Polypeptide chain acquires its secondary and tertiary structure as it emerges from a ribosome. The N-terminal domain folds first, while the C-terminal domain is still being synthesized.

• The protein has not yet achieved its final conformation by the time it is released from the ribosome.

• Mechanisms that monitor protein quality after protein synthesis:1) Correctly fold and assemble protein will be left alone2) Incompletely folded proteins were refolded, with the help

from molecular chaperones: e.g. hsp70, hsp60-like proteins3) Incompletely folded proteins that can not be refold will be

digested by proteosomes4) Combination of all of these processes is needed to prevent

massive protein aggregation in a cell

Action of chaperones during translation

Chaperones bind to the amino (N) terminus of the growing polypeptide chain, stabilizing it in an unfolded configuration until synthesis of the polypeptide is completed.

Protein foldingThe co-translational folding of a protein:The co-translational folding of a protein:- A growing polypeptide chain is shown acquiring its secondary & tertiary structure as it emerges A growing polypeptide chain is shown acquiring its secondary & tertiary structure as it emerges from a ribosome. from a ribosome.

The hsp70 family of molecular The hsp70 family of molecular chaperones:chaperones:Recognize a small stretch of hydrophobic Recognize a small stretch of hydrophobic amino acids on a protein’s surface.amino acids on a protein’s surface.Hsp70 (together with hsp40) binds to it’sHsp70 (together with hsp40) binds to it’s target protein and hydrolyzes a molecule target protein and hydrolyzes a molecule of ATP to ADP, undergoing a conforma-of ATP to ADP, undergoing a conforma-tional change result in hsp70 clampingtional change result in hsp70 clamping tightly to target. Hsp 70 then dissociate tightly to target. Hsp 70 then dissociate induced by rapid induced by rapid re-binding of ATP. re-binding of ATP.

Repeated cycles of hsp Repeated cycles of hsp protein binding & release protein binding & release help the target protein help the target protein to refold.to refold.

hsp70 machinery

hsp70 machinery

Hsp70, 60 & 40

Newly synthesized protein

Correctly folded without help

Correctly folded with help from chaperone

Incompletely folded forms – digested in proteosome

Increasing time

Protein aggregate

The cellular mechanisms that monitor protein quality after protein The cellular mechanisms that monitor protein quality after protein synthesis:synthesis:

• Combination of processes needed to prevent Combination of processes needed to prevent massive aggregationmassive aggregation in a cell, in a cell, which can occur when many which can occur when many hydrophobic regionshydrophobic regions on protein on protein clump togetherclump together and precipitate the entire mass.and precipitate the entire mass.

Different levels of protein structure

• Proteins come in a wide variety of shapes, and generally 50 to 2000 amino acids long

Primary structure: Sequence of amino acids along the core of the polypeptide chain.

Secondary structure: Folding of the polypeptide into most energetically favorable conformation resulting from various non-covalent bonds that form between one part of the chain and another.

Tertiary structure: The full 3-D organization of a polypeptide chain.

Quaternary structure: The highest level of organization, recognized in protein, formed as a complex of more than one polypeptide

Polar and Nonpolar Amino acids

Primary structure

Secondary Structure• Four different noncovalent bonds: ionic bond, H-bond, Van der Waals

attraction & hydrophobic/hydrophilic attraction

Unfolded polypeptide

Folded conformation in aqueous environment

Secondary Structure• Two most common types: -helix & -sheet -helices are held together by H-bond between the N-H and C=O groups

of the polypeptide backbone 4 aa away – Form a regular helix with a complete turn of 3.6 aa

Secondary Structure -sheets are held together by H-bond between the N-H in the peptide

bond on one strand and the C=O of a peptide bond on another sheet strand – Produce a very rigid sheet structure– parallel vs anti-parallel

anti-parallel

parallel

-sheet

Tertiary Structure• Besides the 4 non-covalent bonds, one of the important features is

the formation of disulfide bonds S-S bonds (between cysteines)

cysteine

oxidants

reductantsIntrachain disulfide bond

Interchain disulfide bond

Tertiary Structure

• Protein domain: a substructure produced by any part of a polypeptide chain that fold independently into a compact, stable structure with a specific function, i.e. catalytic domain

SH3 domain

SH2 domain Large kinase domain

Small kinase domain

Quaternary structure• Overall 3D structure assumed by the multimeric protein• Aggregates of more than one polypeptide chain• Individual polypeptide chains that make up multimeric proteins are often

called protein subunits

4o structure of Hemoglobin

Types of protein based on structure:

Globular & Fibrous Globular:

– Tightly folded polypeptide chains, having a much more compact structure.

– Globular proteins include most enzymes and most of the proteins involved in gene expression and regulation, e.g. hemoglobin, deoxyribonucleases, cytochrome c

DNAse

Cytochrome c

Types of protein based on structure• Fibrous:

– Elongated structures, with the polypeptide chains arranged in long strands (parallel strands along a single axis). – Major structural components of the cell or tissue– Examples: collagen (tendon, cartilage, and bone), elastin (skin),

tubulin and actin (cell shape, motility, muscle movement)

Collagen triple helix

Assembly of proteins

• Proteins molecules often serve as a subunits for the assembly of large structure

• Benefits:

– A large structure built from repeating subunits requires smaller amount of genetic information

– Both assembly and disassembly can be readily controlled, reversible processes

– Errors in the synthesis of the structure can be more easily avoided, since correction mechanisms can operate during the course of assembly to exclude malformed subunits

Free subunits Assembled structures

dimer

helix

ring

Proteins • Many proteins have non-peptide components such as

carbohydrate moieties (glycoproteins), metal groups (metalloproteins), lipids (lipoproteins).

• Function of proteins– structure: hair, fingernails.– transport: hemoglobin.– information: protein hormones.– catalysis: enzymes.– locomotion: muscles.

• Protein family: a group of proteins with members having similar - amino acids sequence - three-dimensional (3-D) structure & - function (eg. serine proteases)